Cyclic ADP-ribose (cADPR) is a metabolite of NAD+ that is as effective as inositol trisphosphate in mobilizing intracellular Ca+2 stores in sea urchin eggs (Clapper, D. L., et al., (1987) J. Biol. Chem. 262, 9561-9568; Dargie, P. et al., (1990) Cell Regulation 1, 279-290.) and rat pituitary cells (Koshiyama, H., et al., (1991) J. Biol. Chem. 266, 16985-16988.). The metabolite itself (Walseth, T. F., Aarhus, R., et al., (1991) Biochim. Biophys. Acta 1094, 113-120) as well as its synthesizing enzyme, ADP ribosyl cyclase (Rusinko, N. and Lee, H. C. (1989) J. Biol. Chem. 264, 11725-11731; Lee, H. C. and Aarhus, R. (1991) Cell Regulation 2, 203-209) are present in various mammalian and invertebrate tissues. The cyclic structure of the metabolite is formed by linking the adenine group of NAD+ to the terminal ribosyl unit and displacing the nicotinamide moiety (Lee, H. C., et al., (1989) J. Biol. Chem. 264, 1608-1615). The Ca+2 release mechanism that is activated by cADPR is totally distinct from the inositol trisphosphate pathway. It is insensitive to blockage by heparin, a competitive inhibitor of the receptor for inositol trisphosphate. Furthermore, inositol trisphosphate was shown to complete at least a thousand times less effectively than cADPR for the specific microsomal binding site for cADPR (Lee, H. C. (1991) J. Biol. Chem. 266, 2276-2281).
Several observations indicate involvement of cADPR in the calcium-controlled events taking place during the process of egg fertilization, completion of the oocyte meiotic cycle and the first stages of cell division in invertebrates. Moreover, oscillations of ADP-ribosyl cyclase activity and of cADPR concentration during cell cycle progression have also been reported in the unicellular protist Euglena gracilis. These observations point to an ancient evolutionary role of cADPR as a calcium-releasing signal involved in the control of cell proliferation.
In vertebrates, cADPR has been identified in most tissues and two ADP-ribosyl cyclases, CD38 and BST-1, have been described so far: CD38 is a type II transmembrane glycoprotein, while BST-1 is a glycosyl-phosphatidylinositol-anchored protein. Both are bifunctional ectoenzymes, catalyzing the synthesis and hydrolysis of cADPR at their ectocellular domain. The widespread tissue distribution of cADPR in mammals suggests a role for this nucleotide in calcium-controlled, tissue-specific cell functions, which include secretion, contraction, cell proliferation and apoptosis. Indeed, extracellularly added cADPR has been demonstrated to release calcium from ryanodine-sensitive intracellular stores in a wide range of permeabilized cell types and to elicit tissue-specific functional responses in permeabilized P-pancreatic cells, smooth muscle myocytes and oocytes. cADPR has also been shown to increase cytokine-induced B-lymphocyte proliferation and to increase the depolarization-induced elevation of the intracellular free calcium concentration ([Ca2+]i) in cerebellar neurons.
Recently, xe2x80x9cde novoxe2x80x9d expression of CD38 in CD38xe2x88x92 cells was demonstrated to induce a shortening of the cell cycle, an effect which was shown to be causally related to the intracellular production of cADPR and to the consequent increase of the [Ca2+]i. This observation suggests that cADPR may play a role in the regulation of cell cycle progression.
Currently, there is a need for improved methods and agents useful for promoting the proliferation of hemopoietic progenitor cells without cell differentiation. In particular there is a need for agents that are more potent or more stable to heat and degradation (e.g. enzymatic degradation) than known agents such as cyclic ADP ribose (cADPR). There is also a need for agents that are useful to mobilize intracellular calcium. As well as a need for agents that act as antagonists of cADPR and cADPR induced calcium release.
The present invention provides compounds and methods that are useful for promoting the proliferation of hemopoietic progenitor cells without cell differentiation. Accordingly, the invention provides a compound of formula I: 
wherein:
A is xe2x80x94Nxe2x95x90, or xe2x80x94C(H)xe2x95x90;
D is xe2x80x94C(H)xe2x95x90;
R1 is hydrogen, amino, azido, or halo; and
each R2 is independently hydrogen, or a suitable photolabile caging group;
or a salt or a detectably labeled derivative thereof.
Certain compounds of formula I (e.g. compounds wherein R1 is hydrogen) may be particularly useful to mobilize intracellular calcium. Other compounds of the invention (e.g. compounds wherein R1 is amino, azido or halo) may be particularly useful as stable antagonists of cADPR and cADPR induced calcium release. Representative compounds of formula (I) (e.g. compounds wherein D is xe2x80x94C(H)xe2x95x90) have been found to be more stable to heat and degradation (e.g. enzymatic degradation) than corresponding cADPR analogs.
Additionally, the invention provides a method for promoting the proliferation of a hemopoietic progenitor cell comprising contacting the cell with a compound of the invention.
The invention also provides pharmaceutical compositions comprising a compound of formula I and a pharmaceutically acceptable carrier.
The invention also provides processes and intermediates disclosed herein that are useful for preparing compounds of the invention.
The invention also provides a method to antagonize cADPR induced calcium release in a cell comprising contacting the cell with a compound of formula (I) or a salt thereof.
The invention also provides a method to promote the proliferation of a lymphocyte comprising contacting the lymphocyte with a compound of formula (I) or a salt thereof.
The invention also provides a method to enhance the immune system of a mammal comprising administering to a mammal in need of such treatment, an amount of a compound of formula (I) or a salt thereof.
The invention also provides a method to treat cancer comprising administering to a mammal in need of such treatment, an amount of a compound of formula (I) or a salt thereof.
The invention also provides a method to treat a disease where reduced immune system function is implicated and improved immune system function is desired comprising administering to a mammal in need of such therapy an amount of a formula (I) or a salt thereof.